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Nestin depletion induces melanoma matrix metalloproteinases and invasion.

Lee CW, Zhan Q, Lezcano C, Frank MH, Huang J, Larson AR, Lin JY, Wan MT, Lin PI, Ma J, Kleffel S, Schatton T, Lian CG, Murphy GF - Lab. Invest. (2014)

Bottom Line: Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression.Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood.The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

ABSTRACT
Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated the expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. The development of three-dimensional melanospheres that in vitro partially recapitulate noninvasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase and increased melanoma cell responsiveness to transforming growth factor-beta, both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

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Related in: MedlinePlus

Nestin affects melanoma MMP expression(a) Nestin mRNA levels were significantly down-regulated in nestin KD cells (NesKD, black bars) of A2058 (90% decrease) and A375 (91% decrease,), as compared to vector control cells (Vec, white bars). (b) Decreased nestin protein levels were confirmed in nestin KD A2058 (92.7% decrease) and A375 (97.7% decrease) cells as compared to vector control cells by Western blot. (c) Nestin KD did not significantly alter cell proliferation in A2058 and A375 cells. (d) Compared to A2058 Vector control cells (white bars), A2058 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. e) Compared to A375 Vector control cells (white bars), A375 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. (f) Western blot confirmed that A2058 Nestin KD cells had marked higher protein levels of MMP1 (3.2-fold) and MMP3 (31.2-fold) in cell lysate and secreted MMP1 (3.0-fold), MMP3 (19.9-fold), and MMP10 (60.7-fold) in supernatant. MMP9 protein in supernatant of A2058 cells was below detection limit of immunoblotting. A375 Nestin KD cells had marked higher protein levels of MMP1 (7.3-fold) in cell lysate and secreted MMP1 (4.4-fold) and MMP9 (11.1-fold) in supernatant. MMP3 protein in cell lysate and supernatant of A375 cells were below detection limit of immunoblotting. MMP9 and MMP10 proteins in cell lysate of A2058 and A375 cells were below detection limits of immunoblotting and thus data was not shown.
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Figure 1: Nestin affects melanoma MMP expression(a) Nestin mRNA levels were significantly down-regulated in nestin KD cells (NesKD, black bars) of A2058 (90% decrease) and A375 (91% decrease,), as compared to vector control cells (Vec, white bars). (b) Decreased nestin protein levels were confirmed in nestin KD A2058 (92.7% decrease) and A375 (97.7% decrease) cells as compared to vector control cells by Western blot. (c) Nestin KD did not significantly alter cell proliferation in A2058 and A375 cells. (d) Compared to A2058 Vector control cells (white bars), A2058 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. e) Compared to A375 Vector control cells (white bars), A375 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. (f) Western blot confirmed that A2058 Nestin KD cells had marked higher protein levels of MMP1 (3.2-fold) and MMP3 (31.2-fold) in cell lysate and secreted MMP1 (3.0-fold), MMP3 (19.9-fold), and MMP10 (60.7-fold) in supernatant. MMP9 protein in supernatant of A2058 cells was below detection limit of immunoblotting. A375 Nestin KD cells had marked higher protein levels of MMP1 (7.3-fold) in cell lysate and secreted MMP1 (4.4-fold) and MMP9 (11.1-fold) in supernatant. MMP3 protein in cell lysate and supernatant of A375 cells were below detection limit of immunoblotting. MMP9 and MMP10 proteins in cell lysate of A2058 and A375 cells were below detection limits of immunoblotting and thus data was not shown.

Mentions: We initially produced nestin KD lines in A2058 and A375 cells, one derived from primary skin melanoma (A375), one from lymph node metastasis (A2058) 36. Nestin mRNA levels, averaged over a minimum of four replicate determinations, were significantly downregulated in nestin KD cells of A2058 (89.5% decrease, p<0.001) and A375 (91.4% decrease, p<0.001) lines, as compared to vector controls (Figure 1a). Decreased nestin protein levels were confirmed in nestin KD A2058 (92.7% decrease) and A375 (97.7% decrease) cells as compared to vector control cells by Western blotting (Figure 1b). Nestin KD did not significantly influence overall proliferation in bulk cultures (Figure 1c).


Nestin depletion induces melanoma matrix metalloproteinases and invasion.

Lee CW, Zhan Q, Lezcano C, Frank MH, Huang J, Larson AR, Lin JY, Wan MT, Lin PI, Ma J, Kleffel S, Schatton T, Lian CG, Murphy GF - Lab. Invest. (2014)

Nestin affects melanoma MMP expression(a) Nestin mRNA levels were significantly down-regulated in nestin KD cells (NesKD, black bars) of A2058 (90% decrease) and A375 (91% decrease,), as compared to vector control cells (Vec, white bars). (b) Decreased nestin protein levels were confirmed in nestin KD A2058 (92.7% decrease) and A375 (97.7% decrease) cells as compared to vector control cells by Western blot. (c) Nestin KD did not significantly alter cell proliferation in A2058 and A375 cells. (d) Compared to A2058 Vector control cells (white bars), A2058 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. e) Compared to A375 Vector control cells (white bars), A375 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. (f) Western blot confirmed that A2058 Nestin KD cells had marked higher protein levels of MMP1 (3.2-fold) and MMP3 (31.2-fold) in cell lysate and secreted MMP1 (3.0-fold), MMP3 (19.9-fold), and MMP10 (60.7-fold) in supernatant. MMP9 protein in supernatant of A2058 cells was below detection limit of immunoblotting. A375 Nestin KD cells had marked higher protein levels of MMP1 (7.3-fold) in cell lysate and secreted MMP1 (4.4-fold) and MMP9 (11.1-fold) in supernatant. MMP3 protein in cell lysate and supernatant of A375 cells were below detection limit of immunoblotting. MMP9 and MMP10 proteins in cell lysate of A2058 and A375 cells were below detection limits of immunoblotting and thus data was not shown.
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Related In: Results  -  Collection

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Figure 1: Nestin affects melanoma MMP expression(a) Nestin mRNA levels were significantly down-regulated in nestin KD cells (NesKD, black bars) of A2058 (90% decrease) and A375 (91% decrease,), as compared to vector control cells (Vec, white bars). (b) Decreased nestin protein levels were confirmed in nestin KD A2058 (92.7% decrease) and A375 (97.7% decrease) cells as compared to vector control cells by Western blot. (c) Nestin KD did not significantly alter cell proliferation in A2058 and A375 cells. (d) Compared to A2058 Vector control cells (white bars), A2058 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. e) Compared to A375 Vector control cells (white bars), A375 Nestin KD cells (black bars) had significantly higher mRNA levels of MMP1, MMP3, MMP9, and MMP10. (f) Western blot confirmed that A2058 Nestin KD cells had marked higher protein levels of MMP1 (3.2-fold) and MMP3 (31.2-fold) in cell lysate and secreted MMP1 (3.0-fold), MMP3 (19.9-fold), and MMP10 (60.7-fold) in supernatant. MMP9 protein in supernatant of A2058 cells was below detection limit of immunoblotting. A375 Nestin KD cells had marked higher protein levels of MMP1 (7.3-fold) in cell lysate and secreted MMP1 (4.4-fold) and MMP9 (11.1-fold) in supernatant. MMP3 protein in cell lysate and supernatant of A375 cells were below detection limit of immunoblotting. MMP9 and MMP10 proteins in cell lysate of A2058 and A375 cells were below detection limits of immunoblotting and thus data was not shown.
Mentions: We initially produced nestin KD lines in A2058 and A375 cells, one derived from primary skin melanoma (A375), one from lymph node metastasis (A2058) 36. Nestin mRNA levels, averaged over a minimum of four replicate determinations, were significantly downregulated in nestin KD cells of A2058 (89.5% decrease, p<0.001) and A375 (91.4% decrease, p<0.001) lines, as compared to vector controls (Figure 1a). Decreased nestin protein levels were confirmed in nestin KD A2058 (92.7% decrease) and A375 (97.7% decrease) cells as compared to vector control cells by Western blotting (Figure 1b). Nestin KD did not significantly influence overall proliferation in bulk cultures (Figure 1c).

Bottom Line: Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression.Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood.The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

ABSTRACT
Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated the expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. The development of three-dimensional melanospheres that in vitro partially recapitulate noninvasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase and increased melanoma cell responsiveness to transforming growth factor-beta, both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

Show MeSH
Related in: MedlinePlus